Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes

Abstract

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1 ^Gt/Gt) . Surprisingly, Azi1 ^Gt/Gt MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.

Author Summary

Cilia are slender projections from the surface of most mammalian cells and have sensory and sometimes motile functions. They are essential for mammalian development and defects in cilia lead to a group of human diseases, termed ciliopathies, with variable symptoms including embryonic lethality, lung and kidney defects, blindness and infertility. Cilia are complex structures composed of hundreds of components, whose individual functions are poorly understood. We screened for mammalian genes important in building cilia, and identified Azi1/Cep131, a gene previously shown to be required for cilia formation and function in fish and flies. We show that if we acutely reduce levels of Azi1 in mouse cells, fewer cells form cilia, but if we generate cells chronically lacking all Azi1, cilia form normally. In addition, mice without any Azi1 are healthy and viable, confirming normal cilia function. However, in these mice, the highly specialised ciliary structure of the sperm tail does not form, resulting in male infertility. We suggest Azi1 has conserved trafficking roles in both primary cilia and the specialised sperm flagella. Abruptly removing Azi1 results in instability causing the existing cilia network to collapse, whereas chronic deletion of Azi1 allows the system to re-equilibrate, and cilia to form normally.